There has been considerable interest in the "magic bullet" approach to cancer therapeutics. Recent efforts have been devoted to the conjugation of chemotherapeutic neoplastic drugs to specific antibodies, such as monoclonal antibodies, to produce conjugates which can selectively target tumor cells while sparing normal tissues. Different classes of agents have been considered for this application. These include beta- and alpha-emitting isotopes, plant and bacterial toxins, and a variety of antineoplastic drugs, including intercalating agents, antimetabolites, alkylating agents, and antibiotics. It is desirable to conjugate chemotherapeutic drugs to antibodies for the following reasons:
1. It has recently been shown that up to 1,000-fold more drug can be delivered to tumor cells when conjugated to an antigen-specific monoclonal antibody than is possible by the addition of free drug.
2. Pleiotropic drug resistance may arise following treatment with one of a number of chemotherapeutic drugs, resulting in inducing resistance to drugs of several classes. The mechanism(s) of this resistance are not entirely known, but it is known that this resistance can be partially overcome by antibody targeting of drugs.
3. Even though current chemotherapeutic drugs are active against only some of the major tumor types, the response rate in drug-insensitive tumor types may be increased by antibody-mediated delivery.
4. Many dose-limiting toxicities, which are now seen with chemotherapeutic drugs, may be reduced by conjugation to an antibody. A decrease in toxicity with at least equal efficacy would give a superior product, and the product would have a higher therapeutic index.
To create conjugate with a drug and an antibody, the drug may be directly linked to the antibody through nucleophilic substitution of certain groups on the antibody (e.g., lysines, carboxyl, or sulfhydryl), or the drug may be conjugated to the antibody via hetero- or homo-bifunctional cross-linkers. Linker groups may be small organic compounds or peptides substituted with chemical linkers for conjugation. Large carriers have also been used containing linker groups and offer the advantage of being able to bind many drug molecules to a single antibody. Examples of carriers are the polymers of lysine and glutamic acid, dextran, and the polypeptide albumin.
Drugs have been thus far conjugated to antibodies or carriers only by using covalent bonds. See Biair and Ghose, J. Immunol. Meth. 59:129-144, 1983. Covalent bonds can be further subclassified into non-metabolizable and metabolizable bonds. Metabolizable bonds are those that undergo hydrolysis, releasing the drug under conditions present within or around cells, such as low pH, a reducing environment, or through proteolysis. An example of metabolizable covalent bonds which are useful are those that are sensitive to the low pH environment of endosomes within a cell. See Shen and Ryser, Biochem. + Biophys. Res. Commun. 102: 1048-1054, 1981. After the drug-antibody conjugate binds to the cell, it may be internalized by a pathway that places it in an endosome where the conjugate is subjected to a low pH environment. The hydrolysis of the conjugate's covalent bond releases free drug, where it may then exert its cytotoxic activity.
A non-metabolizable bond can also result in active drug conjugates. However, the resulting conjugates with non-metabolizable bonds generally have reduced drug potency as compared with those conjugates formed with metabolizable bonds. This is because intracellular processing via proteolysis does not release the drug as efficiently as metabolizable bonds. In addition, the drug, if released, is usually in a form different from the native drug and has reduced cytotoxic potency.
Covalent drug-antibody conjugates have been made where the drug is conjugated directly to the antibody and also where the drug is covalently bound to a carrier before conjugation to the antibody. See U.S. Pat. No. 4,507,234, Garnett and Baldwin, Cancer Res. 46: 2407-2412, 1986. Direct conjugation consists of a drug's being conjugated to residues within the antibody molecule, including, for example, lysine and glutamic acid amino groups, sulfhydryl groups and sugar residues within oligosaccharide chains. An important limitation of this direct conjugation approach is that the antibody may be exposed to harsh conjugation conditions, that may denature the antibody causing more rapid clearance from the serum after injection. Unless the direct conjugation is site directed (e.g., at the carbohydrate or sulfhydryl groups), the immunoreactivity of the conjugate may be compromised. Even when combined with site-direction, direct conjugation can still result in nonselectivity (i.e., kill antigen-positive and antigen-negative cells with approximately equal potency) and poor target localization, due to the nature of the agent conjugated to the antibody. As an example, ricin A chain conjugated via site-directed sulfhydryl groups to antibodies is rapidly taken up by liver phagocytes due to mannose receptors for carbohydrate on the ricin A chain. This can also occur with highly lipophilic drugs because lipophilic drugs in free drug form must have some means for interacting with cells to be effective. One such mechanism for lipophilic drugs is insertion into the cell membrane lipid bilayer. If the direct drug-antibody conjugate is formed with a metabolizable covalent bond, this bond can often be metabolized at other sites within the body, such as within the blood, liver, spleen, and other organs.
The indirect method of conjugation first requires the coupling of a drug to a carrier, generally via a linker group. The carrier is then conjugated to the antibody, via a heterobifunctional linker, that can be first conjugated to the carrier and then activated following drug conjugation. One advantage of this indirect conjugation route is that large numbers of drug molecules may be linked to an antibody for delivery to the target site. However, large numbers of drug molecules linked to an antibody may also lead to enhanced nonspecific uptake due, for example, to the lipophilicity of the drug. The indirect conjugation approach does not expose the antibody to the harsh conditions of conjugation, as the chemical manipulations are usually performed on the carrier and not the antibody. Furthermore, the carrier can enhance the solubility of the drug conjugate.
Direct or indirect conjugation of a drug to an antibody creates a stable conjugate that can arrive at the target site with a minimum of dissociation of the drug. One needs, however, to couple this property with a mechanism of selective release of drug for maximal potency.
Selective release may be exploited at three levels. The first is intracellular release within the tumor cell. The best examples of this form of release are pH-sensitive and reducible bonds which, upon intracellular processing of the conjugate, break down to release free drug. This requires binding and internalization of the conjugate prior to drug dissociation. Intracellular internalization of the conjugate requires that the conjugate either enter the cytoplasm or be taken up into an endosome, or lysosome. Internalization rates with monoclonal antibodies to antigens of solid tumors is slow. Thus, a drug conjugate requiring such a process for release of active drug will not be highly potent. In addition, not all internalized conjugate undergoes appropriate intracellular processing for release of active drug. Conjugates that are processed into lysosomes are probably degraded and some drugs will be inactivated. Conjugates processed in the endosomes or into the cytoplasm have the opportunity to release their drugs and allow drugs access to the intracellular target.
A second site for the selective release of drug from the conjugate is the plasma membrane. One example of the plasma membrane release mechanism is referred to in U.S. Pat. No. 4,671,958. In this case, conjugate that is once bound to tumor cells activates complement, which causes the proteolytic degradation of sensitive peptide linkages, to which the drug is bound and releases it in free form.
A third level for drug release would be at the tumor site, but before the conjugate is bound to the tumor cell. This third form of release requires a drug-antibody linkage that would take advantage of certain differences between tumor and normal tissue extracellular milieu. None have been developed to date.
Covalent drug conjugates discussed above comprising cytotoxic or antineoplastic drugs covalently conjugated to an antibody with or without the use of a carrier through linker groups, in a site- or non-site-directed manner, suffer from a number of problems. First, covalent conjugation of drug to antibody requires derivatization of the drug to produce a form of the drug capable of being conjugated to groups in the antibody or carrier. This typically results in a reduction of the drug's cytotoxic activity or potency, due to chemical modification of its functional groups. For some drugs, exposure to the conditions for derivatization may be sufficient to inactivate the drug. For others, the derivatization is not well enough controlled so that groups important for the drug's cytotoxic activity are chemically modified, although these groups are not the primary targets of the procedure. The use of labile bonds, such as pH- sensitive bonds, may overcome part of this problem, but may still result in relatively slow release of the drug at the targeted site or release of the drug in a modified, less active form.
Extracellular release of the drug from the conjugate, as described in U.S. Pat. No. 4,671,958, overcomes the internalization and intracellular processing problems associated with conjugates. The drug, however, still must be derivatized appropriately in order for it to be covalently bound to carbohydrate residues within the antibody molecule, either directly or through a carriermediated system. In addition, the rates of release of the drug will be governed by the half-life of the antibody on the plasma membrane of the tumor cells and by the rate of complement fixation of the antibody. This process is a handicap with most murine monoclonal antibodies (the type most often used), that have little or no ability to fix human complement.
The current generation of immunoconjugates of drugs and antibodies suffer from the additional problem of poor selectivity. This problem of decreased selectivity can be assessed by testing drug conjugates in vitro against antigen-positive and antigen-negative cells. Antigen-positive cells are usually killed at drug-conjugate concentrations tenfold or less lower than antigen-negative cells. This is true, for example, for anthracycline conjugates. Conjugates of the same antibody and a plant or bacterial toxin will, by contrast, typically show 3 to 4 logs of selectivity. It thus seems apparent that the cytotoxic drug itself has additional mechanisms for interacting with cell membranes, and that this leads to nonselective cytotoxicity. Moreover, there is often more than one drug molecule conjugated to an antibody molecule with each drug molecule being capable of nonselective cellular interactions. Thus, there is a considerable need in the art to improve the selectivity of drug immunoconjugates. This can provide improved delivery in vivo to tumor sites as well as decreased normal tissue uptake.